Novel inhibitor peptides II

ABSTRACT

An octapeptide inhibitor of myristoylating enzymes is disclosed having an amino acid sequence as follows or a physiologically acceptable amide or salt derivative thereof: ##STR1##

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of copending application Ser. No.894,185, filed Aug. 7, 1986, now "U.S. Pat. No. 4,709,012".

BACKGROUND OF THE INVENTION

This invention relates to a novel peptide and more particularly to aunique octapeptide which is useful as an inhibitor of myristoylatingenzymes.

Fatty acid acylation of specific eukaryotic proteins is a wellestablished process which can conveniently be divided into twocategories. On the one hand, palmitate (C₁₆) is linked to membraneproteins via ester or thioester linkage post-translationally, probablyin the Golgi apparatus.

On the other hand, it is known that myristate (C₁₄) becomes covalentlybound to soluble and membrane proteins via amide linkage early in theprotein biosynthetic pathway. In the N-myristoylated proteins,amino-terminal glycine residues are known to be the site of acylation.See Aitkin et al., FEBS Lett. 150, 314-318 (1982); Schultz et al.,Science 227, 427-429 (1985); Carr et al., Proc. Natl. Acad. Sci. USA 79,6128-6131 (1982); Ozols et al., J. Biol. Chem. 259, 13349-13354 (1984);and Henderson et al., Proc. Natl. Acad. Sci. USA 80, 339-343 (1983).

The function of protein N-myristoylation is only beginning to beunderstood. Four of the known N-myristoyl proteins --p60^(src), cyclicAMP-dependent protein kinase catalytic subunit, the calcineurinB-subunit, and the Murine Leukemia Virus oncogenic gag-abl fusionprotein-- are either protein kinases or a regulator of a phosphoproteinphosphatase (calcineurin) which modulate cellular metabolic processes.For p60^(v-src), it has been shown that myristoylation is required formembrane association and expression of this protein's cell transformingpotential. See Cross et al., Molec. Cell. Biol. 4, 1834-1842 (1984);Kamps et al., Proc. Natl. Acad. Sci. USA 82, 4625-4628 (1985).

The development of relatively short synthetic peptides which can beconveniently made by synthetic peptide synthesis would be highlydesirable for identifying and in studying the regulation of enzymeaction in fatty acid acylation. Such peptides could serve as syntheticsubstrates for the myristoylating enzyme in yeasts and mammalian cells.They could also serve as highly specific competitive inhibitors of thenaturally-occurring substrates. Novel synthetic peptides which thusserve as substrates of myristoylating enzymes are disclosed in copendingU.S. patent application Ser. No. 924,543, filed Nov. 29, 1986, which isa continuation-in-part of U.S. patent application Ser. No. 894,235,filed Aug. 7, 1986. A preferred example of such substrates is theoctapeptide

    Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg.

Novel synthetic peptides which serve as inhibitors of myristoylatingenzymes are disclosed in co-pending U.S. patent application Ser. No.894,185, filed Aug. 7, 1986. Preferred examples of such inhibitors arethe octapeptides

    Gly-R-Ala-Ala-Ala-Ala-Arg-Arg,

wherein R=Tyr or Phe.

Corresponding octapeptides wherein R=Leu or Val likewise are inhibitorsbut they also serve as substrates of myristoylating enzymes.

The myristoylation reaction can be represented as follows: ##STR2##

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, a novel ppptide inhibitor ofmyristoylating enzymes is provided which has an amino acid sequence asfollows or a physiologically acceptable amide or salt derivativethereof: ##STR3##

An illustrative amide derivatives of this peptide is the carboxyamide.Illustrative salt derivatives are the HCl salts.

The novel octapeptide inhibitor of the present invention which has alysine in position 5 and asparagine in position 2 surprisingly andunexpectedly is about a three-fold better inhibitor than the previouslydescribed inhibitors having alanine in position 5 and tyrosine orphenylalanine in position 2. Furthermore, this novel inhibitor does notalso serve as a substrate of myristoylating enzymes.

DETAILED DESCRIPTION OF THE INVENTION

The novel peptide of this invention can be made by appropriateadaptation of conventional methods for peptide synthesis. Thus, thepeptide chain can be prepared by a series of coupling reactions in whichthe constituent amino acids are added to the growing peptide chain inthe desired sequence. The use of various N-protecting groups, e.g., thecarbobenzyloxy group or the t-butyloxycarbonyl group (BOC), variouscoupling reagents, e.g., dicyclohexylcarbodiimide or carbonyldimidazole,various active esters, e.g., esters of N-hydroxypthalimide orN-hydroxy-succinimide, and various cleavage reagents, e.g.,trifluoracetic acid, HCL in dioxane, boron tris-(trifluoracetate) andcyanogen bromide, and reaction in solution with isolation andpurification of intermediates is well-known classical peptidemethodology.

Preferably, the peptide of this invention is prepared by the well-knownMerrifield solid support method. See Merrifield, J. Amer. Chem. Soc. 85,2149-54 (1963) and Science 150, 178-85 (1965). This procedure, thoughusing many of the same chemical reactions and blocking groups ofclassical peptide synthesis, provides a growing peptide chain anchoredby its carboxyl terminus to a solid support, usually cross-linkedpolystyrene or styrenedivinylbenzene copolymer. This method convenientlysimplifies the number of procedural manipulations since removal of theexcess reagents at each step is effected simply by washing of thepolymer.

The general reaction sequence for conventional Merrifield peptidesynthesis can be illustrated as follows: ##STR4##

Chloromethylation step to provide reactive group for attachment ofpeptide, wherein PS=Polystyrene Residue. ##STR5## EsterificationStep--Reaction with Triethylammonium salt of the First Protected AminoAcid (R¹) Using T-BOC Protecting Group. ##STR6##

Peptide forming step with Dicyclohexylcarbodiimide Coupling Agent.

This step III follows cleavage of t-BOC such as by treatment, forexample, with 25% trifluoracetic acid in methylene chloride andliberation of N-terminal amine by excess of triethylamine, therebyenabling it to react with the activated carboxyl of the next protectedamino acid (R²). A final step involves cleavage of the completed peptidefrom the PS resin such as by treatment, for example, with anhydrous HFin anisole.

Further background information on the established solid phase synthesisprocedure can be had by reference to the treatise by Stewart and Young,"Solid Phase Peptide Synthesis," W. H. Freeman & Co., San Francisco,1969, and the review chapter by Merrifield in Advances in Enzymology 32,pp. 221-296, F. F. Nold, Ed., Interscience Publishers, New York, 1969;and Erickson and Merrifield, The Proteins, Vol. 2, p. 255 et seq. (ed.Neurath and Hill), Academic Press, New York, 1976.

The preferred peptide inhibitor of this invention is

    Gly-Asn-Ala-Ala-Lys-Ala-Arg-Arg.

The preferred octapeptide inhibitor of this invention with lysine inposition 5 and asparagine in position 2 was found to have an apparentK_(i) of 0.05 mM which is a three-fold better level of inhibition thanthe K_(i) of 0.15 mM of the previously described inhibitors with alaninein position 5 and tyrosine or phenylalanine in position 2 and which alsodo not serve as substrates of myristoylating enzymes.

The synthetic octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg was initiallyused to identify a unique enzymatic activity which transfers myristicacid to the amino terminal glycine of this and other peptides. Theinhibitor activity of the novel peptide for the myristoylating enzyme isillustratively demonstrated with the N-myristoylglycylpeptide synthetase(N-myristoyl transferase or NMT) from Saccharomyces cerevisiae. Theenzyme activity was determined in an in vitro assay which measures thetransfer of [³ H]-myristic acid to the acceptor peptide,Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg. The transfer reaction is dependent onadenosine triphosphate (ATP) and coenzyme A (CoA). The enzymatic productwas then identified by high performance liquid chromatography (HPLC) byco-elution with a chemically synthesized myristoyl peptide standard. Todemonstrate that the enzymatic reaction product and the chemicallysynthesized standard were identical and contained myristate covalentlybound to glycine, HPLC-purified standards and enzymatic products wereboth digested with pronase and analyzed by reverse phase HPLC. Bothcontained M-myristoyl glycine.

A protease-deficient strain of Saccharomyces cerevisiae, JR153 [Hemmingset al., Proc. Natl. Acad. Sci. USA 78, 435-439 (1981)], was used as asource of N-myristoylglycylpeptide synthetase to illustrativelydemonstrate the acylation of the octapeptides. This strain was shown tocontain endogenous N-myristoyl proteins by labeling yeast with [³H]myristic acid followed by lysis of cells and analysis of cellularproteins by sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE). N-[³ H]myristoyl glycine could be isolate from labeledendogenous acyl proteins by digestion with pronase followed byseparation and analysis by reversed phase HPLC.

The following examples will illustrate the invention in greater detailalthough it will be understood that the invention is not limited tothese specific examples.

EXAMPLE 1

All peptides herein were prepared essentially by the following methodused for an illustrative peptide as a substrate of myristoylatingenzymes:

A. Synthesis of Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-NH₂

The peptide was synthesized on p-methylbenzhydrylamine resin having asubstitution of 0.35 mmol amino groups/gram of resin by the method ofMerrifield [R. B. Merrifield, J. Am. Chem. Soc., 85, 2149-2154(1963)]BOC-protected amino acids (4 equivalents) were used to formsymmetrical anhydrides by mixing a 2:1 ratio of BOC-amino acid anddicyclohexylcarbodiimide in dichloromethane for 15 minutes. The solventwas evaporated in vacuo and the anhydride was redissolved indimethylformamide and mixed with the resin and agitated for 1 hour. Inthe reaction of asparagine (and glutamine and arginine), an equimolaramount of hydroxybenzotriazole (based on amino acid) was included in thereaction mixture. The BOC-protecting groups were removed using 50%trifluoroacetic acid (TFA) in dichloromethane and the resin wasneutralized with 10% diisopropylethylamine in dimethylformamide prior tocoupling of amino acids.

The peptide was removed from the resin and deprotected using liquidHF/anisole (9:1, v/v) at 0 degrees for one hour. The crude peptide wasextracted from the resin with 50% aqueous acetic acid and lyophilized.

B. Purification

Crude peptide was dissolved in water and applied to a Waters μ-BondapakC₁₈ column (19 mm×150 mm) and eluted with a gradient of 0-15%acetonitrile (0.05% TFA) in water (0.05% TFA) over 15 minutes at a flowrate of 9 ml/min. Fractions containing the product were combined andlyophilized, and the purity and identity of the peptide were ascertainedby analytical HPLC and by amino acid analysis.

EXAMPLE 2 Labeling and extraction of yeast protein for electrophoreticanalysis

Yeast (S. cerevisiae strain JR153, mating type alpha, trpl, prbl, prcl,pep4-3) was grown to an optical density at 660 nm of 1 to 3 in a rotaryshaker at 30° C. in YPD medium (1% yeast extract, 2% Bactopeptone, 2%dextrose in distilled water). Fifteen ml aliquots of yeast culture werelabeled for 30 minutes under identical conditions by addition of 1 mCiof [³ H]fatty acid in 10 μl of ethanol. At the end of the labelingperiod, the cultures were cooled for five minutes on ice and the cellswere pelleted at 4° C. by centrifugation at 7600×g for 10 minutes. Cellswere then resuspended in 1 ml of 10 mM NaN₃ in 140 mM NaCl/10 mMphosphate, pH 7.2, transferred to 1.5 ml polypropylene conicalcentrifuge tubes, and collected by centrifugation at 4° C. as above. Thesupernatant was discarded, and the cells were suspended in 100 μl of 5mM Tris, pH 7.4, 3 mM dithiothreitol, 1% SDS, 1 mMphenylmethylsulfonylfluoride, and broken with one cell volume equivalentof 0.5 mm glass beads, by six 30 second spurts of vigorous vortexingwith cooling on ice between each vortexing. Debris was removed bycentrifugation for 30 seconds at 8000×g in a tabletop Eppendorfcentrifuge. The supernatant was then alkylated in 25 μl of 8 mM Tris, pH8.0, with 20 mM iodoacetamide for 1 hour at room temperature. Twentymicroliter aliquots were analyzed by conventional SDS-PAGE andfluorography methodology essentially as described by Olson et al., J.Biol. Chem. 259, 5364-5367 (1984).

Analysis of the linkage [³ H]fatty acids to proteins

Twenty microliters of the reduced and alkylated [³ H]fatty acid labeledyeast protein was treated with 7 μl of freshly prepared 4Mhydroxylamine/20 mM glycine, pH 10. After treatment for 4 hours at 23°C., samples were prepared for electrophoresis and fluorography as above.

To determine the hydroxylamine-stable linkage of [³ H]myristic acid tothe 20,000 dalton acylprotein in JR153, the cultures were labeled asdescribed above except that the cells were treated for 15 minutes priorto addition of fatty acid with 2 μg/ml cerulenin, a known inhibitor ofyeast fatty acid synthesis which enhances the labeling of the specificacylproteins in JR153 several fold. Cellular protein was then preparedand separated by SDS 12% polyacrylamide gel electrophoresis as describedabove, running molecular weight prestained protein standards in gellanes adjacent to sample lanes. After electrophoresis, 2 mm gel sliceswere cut from the undried gel sample lanes in the 20,000 daltonmolecular weight region. Gel slices were rinsed rapidly with 0.5 ml of10% methanol in water, then individually digested for 72 hours at 37° C.with 1 mg of Pronase E (Sigma, St. Louis, MO) in 1 ml of 50 mM ammoniumbicarbonate, pH 7.9, with mixing on a Labquake mixer (Labindustries,Berkeley, Calif.). One microliter of toluene was added per digest toretard microbial growth. One mg of fresh Pronase E was added at 24hours. Following digestion, the radioactivity present in aliquots ofeach digest was determined. The digest from the slice containingradioactivity was removed, the gel slice was rinsed once with 500 μl of0.1% SDS, and the digest and rinse were combined and acidified to pH 1-2with 40 μl of 6 N HCl. The acidified solution was extracted twice with1.5 ml of chloroform-methanol (2:1, v/v). The combined organic phaseswere backwashed once with 1 ml of chloroform-methanol-0.01 N HCl(1:10:10, v/v/v), and the organic phase dried under a stream ofnitrogen. The residue was redissolved in 50% methanol-50% HPLC buffer A(see below). Ninety-seven percent of the radioactivity present in theoriginal protein digest was recovered after the extraction protocol. Thesample was analyzed by reverse phase HPLC on a Waters μ-Bondapak C₁₈column at a flow rate of 1 ml per min, using as buffer A, 0.1%trifluoroacetic acid/0.05% triethylamine in water, and as buffer B, 0.1%trifluoracetic acid in acetonitrile, eluting with a 1% per minuteacetonitrile gradient. One minute fractions were collected, andradioactivity was determined by liquid scintillation counting. Themyristoyl-[³ H]glycine standard was synthesized essentially as describedby Towler and Glaser, Biochemistry 25, 878-884 (1986), and analyzed byHPLC as above.

Synthesis of fatty acyl peptide standards

The synthesis of acylpeptide standards was performed by reacting theradioactive symmetric myristic acid or palmitic acid anhydride withGlyAsnAlaAlaAlaAlaArgArg in pyridine. One hundred microcuries of [³H]fatty acid was treated with 4 μl of the respective fatty acylchloride, then suspended in 150 μl of pyridine containing 4.8 mg of therespective non-radioactive fatty acid. The reaction was allowed toproceed for 60 minutes at 23° C. Sixty-five microliters of this solutionwas then added to 400-500 μg of GlyAsnAlaAlaAlaAlaArgArg. The reactionwas allowed to proceed overnight with mixing on a Labquake Mixer. Thepyridine was then evaporated under vacuum, the residue extracted twicewith 0.3 ml of petroleum ether, and redissolved in 400 μl of 50%methanol in water. The reaction products were then purified and analyzedby reverse phase HPLC as described above. The chemically synthesizedstandard and the enzymatic product were also both digested with PronaseE and analyzed by reverse phase HPLC as described above for the 20,000dalton acylprotein, except that 200 ug of the protease was sufficientfor complet digestion.

Preparation of yeast extract for the assay of N-myristoylglycylpeptidesynthetase activity

Yeast cultures were grown as described above to O.D. 660 nm of 1 to 3.Cells from 40 ml of culture were collected by centrifugation at 4° C. at7600×g for 10 minutes. The supernatant was decanted, the cell pellet wasresuspended by pipetting into 1 ml of cold 10 mM Tris, pH 7.4,transferred to a 1.5 ml conical polypropylene centrifuge tube, and thecells were then repelleted at 4° C. at 7600×g for 10 minutes. Cells wereresuspended in 400 μl of cold assay lysis buffer (10 mM Tris, pH 7.4, 1mM dithiothreitol, 0.1 mM ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 10 μg/ml Aprotinin) by pipetting.Approximately 400 μl of 0.5 mm glass beads were added to the resuspendedcells, and the cells were lysed by vortexing as described above forlysis of radioactively labeled cells. After allowing the beads tosettle, the lysate was collected and cellular debris was removed bycentrifugation at 4° C. at 1000×g for 10 minutes. The supernatant wasthen centrifuged at 4° C. at 45,000 rpm for 30 minutes in a Beckman 75Ti rotor. The supernatant was removed, and the crude membrane pellet wasresuspended by pipetting into 400 μl of cold assay lysis buffer.Aliquots of the three cellular fractions were either assayed immediatelyor stored at -60° C. The activity associated with crude membranes wasstable at -60° C. for at least 3 months. Protein was determined by themethod of Peterson, Anal. Biochem. 83, 346-356 (1977).

Assay for N-myristoylglycylpeptide synthetase activity

[³ H]Fatty acyl CoA was synthesized enzymatically and added to theincubation as follows. The acyl CoA synthetase reaction consisted of(per one assay tube): 0.5 μCi of [³ H]myristic acid; 25 μl of 2X assaybuffer (20 mM Tris, pH 7.4, 2 mM dithiothreitol, 10 mM MgCl₂, 0.2 mMEGTA); 5 μl of 50 mM ATP in distilled water, adjusted to pH 7.0 withNaOH; 2.5 μl of 20 mM lithium CoA in distilled water; 15 μl of lmU/μl ofPseudomonas acyl CoA synthetase (Sigma) in 50 mMN-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid, pH 7.3; 2.5 μl ofdistilled water. The reaction was allowed to proceed for 20 minutes at30° C. Typically, 40% to 50% of the [³ H]fatty acid was converted to itsCoA ester by this procedure as measured by determining the radioactivityremaining in the reaction after acidification with 6 N HCl to pH 2.0 andextraction 6 times with 5 volumes of heptane, a modification of themethod of Hosaka et al., Meth. Enzymol. 71, 325-333 (1981). Fiftymicroliters of this reaction were added to tubes containing 40 μl ofassay extraction buffer (see above) and 10 μl of 1 mMGlyAsnAlaAlaAlaAlaArgArg. The assay was initiated by the addition of 10μl of yeast cell extract (typically 50 μg of protein) per tube, followedby incubation at 30° C. for 10 min. The assay was terminated by theaddition of 110 μl of methanol and 10 μl of 100% trichloracetic acid(w/v) per tube, followed by cooling ten minutes on ice. Precipitatedprotein was removed by centrifugation at 8000×g for 3 minutes in atabletop Eppendorf centrifuge. (Under these conditions, 95% of synthetic[³ H]myristoylpeptide or [³ H]palmitoylpeptide remined soluble whenadded to an assay mixture.) Fifty microliters of the supernatant weremixed with 75 μl methanol and 75 μl of HPLC buffer A, and analyzed byreverse phase HPLC on a 3.9 mm by 30 cm Waters μ-Bondapak C₁₈ columnusing the same HPLC buffers described above, starting at 35%acetonitrile and eluting with a 1% per minute acetonitrile gradient. Oneminute fractions were collected and the radioactivity in each fractiondetermined by liquid scintillation counting. [³H]Myristoyl-GlyAsnAlaAlaAlaAlaArgArg eluted at 24 minutes, while [³H]palmitoyl-GlyAsnAlaAlaAlaAlaArgArg eluted at 30 minutes.

Results

The chemically synthesized standards of [³ H]-myristoylglycylpeptide and[³ H]palmitoylglycylpeptide prepared as described above were found toelute from the reverse phase HPLC column with 59% and 65% acetonitrile,respectively, under the conditions used for analyzing assay samples. Inthe cell lysates prepared and fractionated into crude membranes andsoluble fractions above, N-Myristoylglycylpeptide synthetase activitywas detected in both crude membrane and soluble fractions, with thespecific activities of total, soluble, and membrane fractions being1410, 1320, and 2260 dpm per μg protein per 10 min assay, respectively.From the initial reaction velocities, it was estimated that 65% of theactivity resided in the crude membrane fractions.

The enzymatic reaction product and the chemically synthesized standard[³ H]-myristoylpeptide were demonstrated to be identical and to containmyristate covalently bound to glycine when analyzed by the reverse phaseHPLC as described above.

To demonstrate the specificity of the N-myristoylglycylpeptidesynthetase for the peptide substrate, the ability of otherglycylpeptides to competitively inhibit acylation ofGlyAsnAlaAlaAlaAlaArgArg also was examined. As can be seen in Table I,below, Test 3, 1 mM concentrations of a dipeptide, a tetrapeptide, and adecapeptide had no effect on myristoylation of 18 μM peptide substrate(ca. one-eighth its K_(m)). Thus, the N-myristoylglycylpeptidesynthetase exhibits specificity for the octapeptide substrate.

                  TABLE I                                                         ______________________________________                                        Characterization of N--myristoylglycine peptide synthase                                             Rate of Myristoylpeptide                                                      Synthesis                                              Test                   DPM × 10.sup.-3 /10 min                          ______________________________________                                        1      Control         111                                                           -ATP            9                                                             -CoA            1                                                      2      Control         83                                                            Heated Membranes                                                                              2                                                             (5 min/65°)                                                     3      Control         26.7                                                          +1 mM GN        28.0                                                          +1 mM GPRP      25.6                                                          +1 mM GSSKSPKDPS                                                                              27.4                                                   ______________________________________                                    

Assays were carried out as described above, using crude membranefractions from yeast with changes as indicated. In Test 1, thedependence of the assay on ATP and CoA was tested in the absence ofexogenous fatty acid CoA ligase. In Test 2, it was demonstrated that theyeast enzyme is heat labile, and in Test 3, that addition of otherpeptides containing N-terminal glycine does not inhibit the reactionwhich in this test was measured using only 18 μM peptide substraterather than the usual 90 μM, in order to maximize possible inhibitoryeffects.

EXAMPLE 3

An octapeptide illustrating the present invention and several otheroctapeptide inhibitors used for comparative purposes were synthesized bythe solid phase Merrifield procedure essentially as described in Example1 and then tested for activity as substrates or inhibitors for themyristoylating enzyme from yeast (S. cerevisiae strain JR153). Theoctapeptide substrate specificity of the enzyme was tested under theassay conditions described in Example 2 but using 1 μCi of [³ H]-myristic acid per assay tube. The yeast enzyme used in this example waspartially purified from a crude homogenate of the cultured yeast cellsby fractionation with 51-70% (NH₄)₂ SO₄ followed by ion exchange columnchromatography with DEAE-Sepharose® CL-6B (Pharmacia) and affinitychromatography with CoA-agarose affinity matrix (Pharmacia). Theoctapeptides were characterized kinetically with the respective kineticdata (K_(m), V_(max) and K_(i)) being shown in Table II below.

                                      TABLE II                                    __________________________________________________________________________    Octapeptide Substrate Specificity of Yeast                                    Myristoylating Enzyme                                                                                Relative                                                                      K.sub.m                                                                            V.sub.max (%)                                                                      K.sub.i                                      Octapeptide Sequence   (mM)      (mM)                                         __________________________________________________________________________    Gly--Asn--Ala--Ala--Ala--Ala--Arg--Arg                                                               0.06 100*                                              Gly--Val--Ala--Ala--Ala--Ala--Arg--Arg                                                               0.7  8    0.06                                         Gly--Leu--Ala--Ala--Ala--Ala--Arg--Arg                                                               0.3  5    0.06                                         Gly--Tyr--Ala--Ala--Ala--Ala--Arg--Arg                                                                         0.15                                         Gly--Phe--Ala--Ala--Ala--Ala--Arg--Arg                                                                         0.15                                         Gly--Asn--Ala--Ala--Lys--Ala--Arg--Arg                                                                         0.05                                         __________________________________________________________________________     *The V.sub.max for this octapeptide substrate was 2840 pmol myristoyl         peptide formed per minute per mg of the partially purified yeast enzyme. 

The foregoing results were unexpected and surprising insofar as theyshow that the octapeptide with lysine in position 5 and asparagine inposition 2 was a competitive inhibitor of NMT while the octapeptide withalanine in position 5 was a substrate. Furthermore, the novel inhibitorhad a three-fold better inhibition constant (K_(i)) than the octapeptideinhibitors with alanine in position 5 and tyrosine or phenylalanine inposition 2 which also did not serve as a substrate of myristoylatingenzymes.

Standard amino acid abbreviations are used to identify the sequence ofthe peptides herein as follows:

    ______________________________________                                        Amino Acid           Abbreviation                                             ______________________________________                                        L-Alanine            Ala or A                                                 L-Arginine           Arg or R                                                 L-Asparagine         Asn or N                                                 L-Aspartic acid      Asp or D                                                 L-Glutamine          Gln or Q                                                 L-Glycine            Gly or G                                                 L-Leucine            Leu or L                                                 L-Lysine             Lys or K                                                 L-Phenylalanine      Phe or F                                                 L-Proline            Pro or P                                                 L-Serine             Ser or S                                                 L-Tyrosine           Tyr or Y                                                 L-Valine             Val or V                                                 ______________________________________                                    

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention, and it is intended that all such otherexamples be included in the scope of the appended claims. Thus,variations in the individual amino acids and/or the chain length of thepeptides which do not adversely or detrimentally affect their biologicactivity as inhibitors for myristoylating enzymes as defined herein areintended to be included within the scope of the appended claims.

What is claimed is:
 1. An octapeptide inhibitor of myristoylating enzymes having an amino acid sequence as follows or a physiologically acceptable amide or salt derivative thereof: ##STR7## 